Regulation of many immune response genes depend on a 10 bp DNA sequence termed kappaB. This sequence is bound by a family of protein factors related to the Rel oncogene. The prototype transcription complex binding to the sequence, termed NF-kappaB, has been conventionally defined as a heterodimer between a P50 DNA binding protein and a P65 (RelA) activation protein that is typically sequestered in the cytoplasm by a protein called I-kappaB. Following certain types of stimulation to the cell, a specific protein kinase complex called I-kappaB kinase causes the phosphorylation of I-kappaB followed by its ubiquitination and degradation. Among the stimuli that can release NF-kappaB is the triggering of the T cell receptor (TCR) or B cell receptor (BCR) by antigen during an immune response. While studying a rare clinical condition of immunodeficiency, we discovered that NF-kappaB has a more complex subunit composition than previously suspected. Specifically, we found that genes such as IL-2 and I-kB harbor kappaB sites of a particular sequence that bind a trimeric complex containing p50, p65, and ribosomal protein small subunit 3 (RPS3). RPS3 is a K-homology (KH) domain-containing protein that binds single-stranded nucleic acids and dramatically enhances the affinity of the p50 and p65 Rel-homology proteins to these select kappaB sites. A subset of all NF-kappaB genes are dependent on RPS3 including crucial physiological functions such as expression of the immunoglobulin kappa light chain gene in B cells and the interleukin-2 gene in T cells. We have further explored this new paradigm for NF-kappaB gene regulation to potentially explains the selective activation of genes harboring distinct versions of NF-kappaB binding sites. This has led us to search for other subunits that may also participate in the binding complex. We have determined that there is a distinct KH domain protein called wan23 is a critical subunit of the NF-kappaB complex that binds to the particular kappaB binding site found in the promoter of the CD25 (the IL-2 receptor alpha chain) gene. DNA-binding analyses reveal that wan23 dramatically enhances the binding of the p50 and p65 Rel subunits to the CD25 promoter site. Moreover, the transcriptional activity of this site mediated by NF-kappaB is profoundly suppressed if wan23 is depleted by siRNA. In functional analyses, we have determined that the participation of wan23 in NF-kappaB-mediated gene regulation in human leukemia cell lines governs the survival of these cells in response to interleukin-2 in cell culture. If we reduce the expression of wan23, various human leukemia cell lines such as Hut-102 or MT-2 have markedly reduced viability. Therefore, wan23 is a critical physiological component of particular NF-kappaB regulatory complexes. This has verified our hypothesis that there are a group of nuclear NF-kappaB complexes that have distinct subunit compositions that have previously been viewed as a single NF-kappaB complex. These discoveries elucidate a fundamental mechanism by which NF-kappaB mediates specific gene regulatory effects. Further work is being directed to understanding how the RPS3 and wan23 KH domain proteins are regulated to become incorporated into the NF-kappaB complexes at specific genes. Preliminary analyses suggest that specific phosphorylation events play a key role in this process. We are also attempting to identify inhibitors of this component, since inhibition of NF-kappaB is a prime therapeutic target for number of inflammatory and degenerative conditions. Inhibition of NF-kappaB may also be useful for various infectious diseases involving pathogens, such as HIV, that utilize this factor for their life cycle or pathogenic effects. In particular, we are studying the subunit composition of the NF-kappaB complex that binds to the kappaB sites in the HIV long terminal repeat transcriptional promoter. We have also been studying the activation circuitry that induces NF-kappaB after triggering the antigen receptor on B cells or T cells. We have found a new kinase that plays a direct role in physically linking the membrane-associated protein complex containing the Carma 1, MALT 1, and the Bcl-10 (CBM) proteins to the I-kappaB kinase complex. We have demonstrated a vital role for the cellular kinase in the induction pathway. This novel kinase has both a positive and negative regulatory role is transducing the signals from antigen receptors at the surface of lymphocytes to the gene induction apparatus. This kinase has been shown to be involved in developmental and circadian rhythm pathways and now appears to play a key role in immune function. We will be studying the biochemical features of its regulation to understand how it might be modulated in various diseases of the immune system. Mass spectrometry analysis has also revealed other proteins in the signal transduction pathway involving the CBM complex and experimentation will be directed to elucidating the functional role of these proteins.